1. Field of the Invention
The present invention relates to the field of cancer and to the field of evaluation of therapy of diseases, including glioblastoma, and the use of alkylating agents and other therapeutics.
2. Related Art
The prognosis of patients with glioblastoma multiforme has not improved substantially over the past decades and almost all patients succumb to their disease. Current treatment approaches are based on radiation therapy and alkylating agent chemotherapy. O6-guanine alkylating agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ), are among the most widely used chemotherapeutics in treating glioblastomas because they can efficiently cross the blood-brain barrier. These agents have modest efficacy against glioblastomas (Refs 1, 2). A subset of glioblastoma patients demonstrates an initial response, lasting a few or several months and eventually leading to tumor recurrence.
One of the most prominent resistance mechanisms to alkylating agents includes O6-methylguanine DNA methyltransferase (MGMT) (Ref 3), which acts as a suicide enzyme by removing the methyl or chloroethyl damage at the O6-position of guanine. Epigenetic MGMT gene silencing via promoter hypermethylation, present in about 40% of cases, has been shown to predict outcome in glioblastoma patients treated with BCNU or TMZ (Refs. 4, 5).
The growing awareness that resistance in human cancer is likely regulated by the coordinated alteration of molecular pathways (Ref. 6) suggests that many more genes might be involved in the development of resistance phenotypes in glioblastomas than the changes described thus far for a limited number of known resistance genes (Ref. 3). Resistance of glioblastomas to alkylating agents such as BCNU and TMZ seems to follow a more complex pattern than simple dependence on MGMT levels (Refs 3,7-9).
Excessive and prolonged activation of nuclear factor-κB (NF-κB) has been established as a principal mechanism of tumor chemoresistance, which is primarily mediated by its antiapoptotic activity (Refs. 10,11). Some evidence also indicates a link between the NF-κB pathway and resistance of glioblastoma cells to O6-alkylating agents, and suggests that inhibition of NF-κB is a promising means to potentiate the cytotoxic effects of these agents (Ref. 12). The NF-κB complex consists of a family of heterodimers, of which the p50/p65 heterodimer is the most abundant form. NF-κB is active in the nucleus and is inhibited through its sequestration in the cytoplasm by the inhibitors of κB (IκB), primarily through the interaction of IκB proteins with p65. IκB is a target of several well-characterized kinase cascades that activate IκB kinases (IKK), which phosphorylate IκB and mark it for degradation via the ubiquination pathway, thereby allowing activation of NF-κB. Activated NF-κB translocates to the nucleus and binds DNA at κB-binding motifs, which initiates gene transcription. Anticancer drugs are known to induce the expression of NF-κB target genes through the direct activation of NF-κB and the secondary production of NF-κB activators (Ref 11).
There is increasing recognition of the value of comprehensive approaches to the molecular characterization of biological phenotypes such as drug resistance. We have here utilized an integrated model of glioblastoma resistance to O6-alkylating agents and genomics tools to globally explore molecular factors, cellular pathways, and functional interaction networks perturbed during the selection and evolution of drug resistance in glioblastoma cells.
The results described below highlight the involvement of a cellular pathway of NF-κB-mediated resistance to these agents in glioblastoma cells. The contribution of NF-κB to anticancer drug resistance has been described in various in vitro and in vivo resistance models (Ref. 10). The antiapoptotic activity of NF-κB appears to be the most important mode of action mediating the resistance and pro-survival effects of this gene in cancer cells (Refs. 10-13). Genotoxic stress resulting from the exposure of tumor cells to O6-alkylating agents causes DNA damage and leads to the initiation of apoptosis. NF-κB activation abrogates the apoptosis signal in response to these agents (Ref. 10). Antagonism of NF-κB in malignant gliomas has been shown to render glioma cells more susceptible to BCNU via increased apoptosis (Ref. 12), but the DNA damage-induced signaling pathway upstream of IκB has not been identified in these cells.
Cited Patents and Publications
Inactivation of the DNA-Repair Gene MGMT and the Clinical Response of Gliomas to Alkylating Agents, by Esteller, M, et al., N Engl J Med. 2000; 343(19): 1350-1354 describes how the DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents. MGMT activity is controlled by a promoter; methylation of the promoter silences the gene in cancer, and the cells no longer produce MGMT. The authors examined gliomas to determine whether methylation of the MGMT promoter is related to the responsiveness of the tumor to alkylating agents. They found that the MGMT promoter was methylated in gliomas from 19 of 47 patients (40 percent). This finding was associated with regression of the tumor and prolonged overall and disease-free survival. It was an independent and stronger prognostic factor than age, stage, tumor grade, or performance status. The authors concluded that methylation of the MGMT promoter in gliomas is a useful predictor of the responsiveness of the tumors to alkylating agents.
US 2005/0287541 to Nakagawara, et al., published Dec. 29, 2005, entitled “Microarray for predicting the prognosis of neuroblastoma and method for predicting the prognosis of neuroblastoma,” discloses microarray for predicting the prognosis of neuroblastoma, wherein the microarray has 25 to 45 probes related to good prognosis, which are hybridized to a gene transcript whose expression is increased in a good prognosis patient with neuroblastoma and are selected from 96 polynucleotides.
US 2003/0198961 to Spelsberg, et al., published Oct. 23, 2003, entitled “Determining cancer aggressiveness,” discloses methods for determining the aggressiveness of a cancer in a mammal. Specifically, the invention provides methods and methods for measuring the level of a TIEG marker in a sample. Such levels can be correlated with the aggressiveness of a cancer to predict patient outcome and develop treatment regimens.
A list of additional cited references is contained at the end of the specification.